Inhibitor of tissue factor activity

ABSTRACT

A sulfated glycoprotein with a molecular weight of approximately 45 kda inhibits the activation of tissue factor and thus inhibits the coagulation of blood. This glycoprotein can be used for treatment or prevention of intravascular clotting.

This is a division of application Ser. No. 07/830,462, filed Feb. 5,1992, now U.S. Pat. No. 5,219,994, which is a continuation ofapplication Ser. No. 07/707,314, filed May 29, 1991, now abandoned,which is a continuation of application Ser. No. 07/268,893, filed Nov.8, 1988, now abandoned.

FIELD OF THE INVENTION

The present invention is directed to a method and composition forinhibiting the activity of tissue factor.

BACKGROUND OF THE INVENTION

Maintenance of vascular integrity is an important host response toinjury- Complex hemostatic mechanisms of coagulation, platelet function,and fibrinolysis exist to minimize adverse consequences of vascularinjury and to accelerate vascular repair. Many of these hemostaticmechanisms are initiated and/or regulated by cells of the wall of theblood vessel.

A number of plasma proteins and circulating factors in blood areinactive until the events that stimulate clotting are triggered, atwhich point the various factors involved in clot formation areactivated. Several different types of activation are to be considered incoagulation. One type is the activation of a zymogen of a proteolyticenzyme, the product of which, in turn, activates another zymogen byproteolytic action. Another type does not lead to activation of anenzyme. For example, the proteolytic cleavage of a soluble plasmaprotein, fibrinogen, results in formation of fibrin, the insolubleprotein that forms the clot. Similarly, platelets, the cells that areessential for normal coagulation, must also be activated before they canparticipate in coagulation.

Classically, the initiation of blood clotting can be conceptuallyseparated into two different, yet similar, molecular mechanisms, calledthe intrinsic and extrinsic coagulation pathways or cascades. Themechanism of initiation pertaining to this patent application is theextrinsic coagulation pathway. The two pathways converge at a step, theactivation of prothrombin to thrombin by factor X_(a), prior to theactual clot formation. There is also some feedback regulation betweenthe two systems. In the extrinsic pathway, for optimal conditions, eachreaction of the coagulation mechanism involves assembly on a cellularsurface of a complex that includes a serine protease, a substratezymogen, and a nonenzymatic cofactor protein, with or without calciumions. Complex formation on the cell surfaces localizes the hemostaticresponse, promotes optimal activation of coagulation, and protectsproteases from their plasma inhibitors.

The actual set of cascade reactions of the extrinsic pathway are asfollows: in the presence of calcium, found in circulation, tissuefactor, a membrane-bound procoagulant enzyme, combines with factor VII,a circulating proenzyme, and the complex is activated to TF-VII_(a) byeither X_(a), which can be found in very low concentrations in thecirculating blood, or by high molecular weight kallikrein acting onXII_(a) of the intrinsic pathway, also found in the circulation. Oncethe complex has been activated it can active X, a proenzyme in blood, toX_(a), a serine protease. X_(a) can now activate, in the presence offactor V, (a cofactor found in circulation), prothrombin to thrombin.Thrombin will then cleave fibrinogen to form fibrin.

Factor V and tissue factor can be synthesized by vascular endothelium,whereas endothelial cells posses binding sites for the plasma proteins,and high molecular weight kininogen, allowing expression of thesecoagulant activities.

Once thrombin is generated, fibrinogen is cleaved to form fibrin, whichis substantially cross-linked by factor XIII_(a) to form an insolublefibrin clot. Enmeshed in the clot are platelets that have been recruitedto the site of vascular injury after exposure to subendothelialcomponents such as collagen.

When vascular cells are exposed to perturbing stimuli, a number ofcellular hemostatic properties are altered, including increasedexpression of procoagulant activity, decreased expression ofanticoagulant activity, and enhanced platelet adhesion and activation.

The coagulation mechanisms can be initiated either by expression oftissue factor activity, (extrinsic pathway), or by activation of factorXII, (intrinsic pathway).

A number of stimuli have been found to induce tissue factor activity.These stimuli include immune modulators such as immune complexes, themonokines interleukin 1 and tumor necrosis factor, other stimuli linkedto infection such as endotoxin and microorganisms associated withbacterial endocarditis, a metabolite associated with thrombotic disease(homocysteine), and other stimuli such as mechanical injury and phorbolesters. With the exception of endothelial cell injury induced bymechanical means or by gross infection (such as endocarditis), theinduction of these vascular coagulant properties is not normallyassociated with overt cell injury.

SUMMARY OF THE INVENTION

It is an object of the present invention to inhibit tissue factoractivity.

It is another object of the present invention to provide an endothelialcell glycoprotein that inhibits the initiation of the coagulationprocess promoted by tissue factor.

It is a further object of the present invention to provide a method tomaintain blood fluidity.

It is a further object of the present invention to provide compounds foranticoagulant therapy.

It is yet another object of the present invention to provide compoundsfor prevention and treatment of intravascular clotting.

An endothelial cell glycoprotein that inhibits the initiation of thecoagulation process promoted by tissue factor has been identified. Thisglycoprotein was isolated by heparin-sepharose, hydroxyapatite, and gelfiltration chromatography. The N-linked carbohydrate moiety is sulfated.

The endothelial cell product of the present invention is one of theseveral N-glycan sulfated glycoproteins synthesized by endothelialcells. This molecule has an apparent molecular weight of 45 kDa, havinga partial sequence of 22 amino acids, 19 of which were positivelyidentified.

As used herein, the term "salts" includes both salts of carboxyl groupsand to acid addition salts of amino groups of the protein molecule.Salts of a carboxyl group may be formed by means known in the art andinclude inorganic salts, for example, sodium, calcium, ammonium, ferric,or zinc salts, and the like, and salts with organic bases such as thoseformed, for example, with amines, such as triethanolamine, arginine, orlysine, piperiodine, procaine, and the like. Acid addition saltsinclude, for example, salts with mineral acids such as, for example,hydrochloric acid or sulfuric acid, and salts with organic acids suchas, for example, acetic acid or oxalic acid.

Functional derivatives of the glycoproteins according to the presentinvention include derivatives which may be prepared from the functionalgroups which occur as side chains on the residues or the N-0 orC-terminal groups, by means known in the art, and are included in theinvention as long as they remain pharmaceutically acceptable, i.e., theydo not destroy the activity of the protein and do not confer toxicproperties on compositions containing it.

These derivatives may include, for example, aliphatic esters of thecarboxyl groups, amides of the carboxyl groups by reaction with ammoniaor with primary or secondary amines, N-acyl derivatives of free aminogroups of the amino acid residues formed with acyl moieties (e.g.,alkanoyl or carbocyclic aroyl groups) or O-acyl derivatives of freehydroxyl groups (for example, that of seryl or threonyl residues) formedwith acyl moieties.

As "active fractions" of the substantially purified protein, the presentinvention covers any fragment or precursors of the polypeptide chain ofthe protein molecule alone or together with associated molecules orresidues linked thereto, e.g., sugar or phosphate residues, oraggregates of the protein molecule or the sugar residues by themselves,provided that said fraction has the ability to inhibit the activity oftissue factor.

One probable amino acid sequence for the inhibitor of the presentinvention, wherein the positively identified amino acids are noted inbold type, is as follows, wherein X represents unidentified amino-acids:

NH₂-X-X-Glu-Glu-Asp-Glu-Glu-Phe-Thr-X-Ile-Thr-Asp-Ile-Lys-Pro-Pro-Leu-Gln-Lys-Pro-Thr-BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B identify the endothelial cell inhibitor of the presentinvention. FIG. 1A shows gel filtration chromatography of thehydroxyapatite eluate, and FIG. 1B shows the gel electrophoresis ofvarious materials isolated.

FIG. 2 shows inhibition of factor X activation by the endothelial cellproduct according to the present invention.

FIGS. 3A and 3B are graphs which show how the addition of increasingamounts of tissue factor overrides the block by the endothelial cellinhibitor.

DETAILED DESCRIPTION OF THE INVENTION Isolation of the Endothelial CellInhibitor

A cell line from the intima of the rabbit aorta was establishedaccording to the procedure of Buonassisi et al. as reported in Proc.Natl. Acad. Sci. USA 73: (5) 1612-1616, 1976, which article is hereinincorporated by reference. This cell line exhibits strict contactinhibition and morphologically resembles intimal endothelial cells. Thiscell line possesses B-type blood group antigens and fibrinolyticactivity. Norepinephrine, acetylcholine, 5-hydroxytryptamine, andphenylephrine increased the levels of both cAMP and cGMP significantly.Propanolol and phentolamine inhibited, respectively, the cAMP and cGMPresponse to norepinephrine. Angiotensin II and histamine significantlyincreased cGMP levels but not cAMP levels of the endothelial cells. ThecGMP increases with acetylcholine were inhibited by atropine.

conditioned medium, 500 ml, from cell cultures labelled with H₂ ³⁵ SO₄was applied to a 1.5×2 cm heparin-Sepharose column. The fractionscontaining the inhibitory activity, which was eluted with 0.45 M NaClwith a linear NaCl gradient (0.15 M to 1.15 M in 0.02 M sodium phosphatebuffer, pH 7.4), were applied to a 1.5 ×1.5 cm hydroxyapatite column.The material present in the bypass was subjected to electrodialysisconcentration and fractionated on a 1.5×60 cm Sephacryl S-300 columnusing as an eluant 0.5 M NaCl in 0.02 M sodium phosphate buffer, pH 7.4.The inhibitor was further purified on a preparative 10%SDS-polyacrylamide gel slab and eluted from the gel by electrodialysis.

The inhibitory activity was estimated with an assay system that includedfactors VII and X and a chromogenic substrate specific for factor X_(a): 20 μl of sample was added to 160 μl solution containing 0.05 unitsfactor VII, 0.1 units factor X, and 50 μg bovine serum albumin in 0.02 Msodium phosphate, 0.9% NaCl, pH 7.4. The reaction was initiated by theaddition of 40 μl solution containing 22 μl thromboplastin (Sigma,prepared according to instructions from the manufacturer, as a source oftissue factor), 4 μl S-2222 (7 mM), and 14 μl 25 mM CaCl₂. Theabsorbancy at 405 nm was measured over time. The slope of the reactionwas determined using the time points where the absorbance increaselinearly.

Assay for the Inhibition of Factor X Activation

Five hundred microliters of the incubation mixture contained 0.16 unitsfactor VII, 0.31 units factor X, in buffer (Earle's balanced saltsolution), and 60 μl of inhibitor concentrate (hydroxyapatite bypass).The control received 60 μl buffer. Fifty microliters of thromboplastinsolution (25 μl thromboplastin in 200 μ25 mM CaCl₂) was added toinitiate the reaction. The solutions were left at room temperature. Atthe times indicated, 100 μl was removed from each tube, placed inseparate tubes containing 50 μl sample buffer (10% SDS, 20%2-mercaptoethanol, 30% glycerol) and boiled for five minutes. Fiftymicroliters were analyzed by analytical SDS-polyacrylamide gel. Afterelectrophoresis, the proteins were transferred to nitrocellulose, andfactor X and its fragments were detected using a rabbit anti-humanfactor X-peroxidase antibody.

Site of Inhibition of the Coagulation Pathway

For the experiments described herein, the methodology was as follows:Ten microliters of inhibitor (hydroxyapatite bypass) was added to tubescontaining 150 μl non-conditioned medium supplemented with 10% fetalbovine serum as a source of clotting factors and increasing amounts offactor VII. Control samples contained 10 μl buffer in place of theinhibitor. The reaction was initiated with the addition of 20 μlthromboplastin-substrate solution: 25 μl thromboplastin, 25 μl S-2238(15 mM, a substrate specific for thrombin), 200 μl 25 mM CaCl₂. Theamount of thromboplastin present in this reaction mixture was adjustedfor the various samples when the experiments requiring increasingamounts of tissue factor were performed.

Similar results are obtained when the assays are performed with purifiedfactors. In the experiments described herein, the reaction rate wasstrictly dependent on the addition of factor VII and was the same incontrol samples containing zero units of factor VII as in the samplescontaining the inhibitor.

The molecule responsible for the inhibitory activity is a species thatcan be initially concentrated on a heparin-Sepharose column and which isfurther purified by hydroxyapatite and gel filtration chromatography.This species can be finally isolated in a highly active state and freeof other proteins by polyacrylamide gel electrophoresis, as shown inFIGS. 1A and 1B. This endothelial cell product is one of the severalN-glycan sulfated glycoproteins synthesized by this cell type.

To identify the endothelial cell inhibitor of the present invention, asshown in FIGS. 1A and 1B, the molecule was purified from ³⁵ S-labelledconditioned medium by heparin-Sepharose, hydroxyapatite, and gelfiltration chromatography- The material present in the peak of activityfrom the gel filtration column (which contained only one ³⁵ S-labelledspecies) was subjected to preparative polyacrylamide gelelectrophoresis. The active material was eluted from the gel slab, andan aliquot was rerun on an analytical gel. In FIG. 1A, the gelfiltration chromatography of the hydroxyapatite eluate is as follows: --, ³⁵ S-radioactivity; □--□, inhibitory activity. FIG. 1B shows thereducing, analytical polyacrylamide gel electrophoresis of 1) materialpresent in the peak of inhibitory activity isolated by gel filtration(silver stain); 2) active material eluted from the preparative gel(silver stain); 3) the same material visualized by autoradiography; 4)autoradiography of the ³⁵ S-labelled species present in the fractionscontaining inhibitory activity from the heparin-Sepharose column beforetreatment with N-glycanase (an enzyme that splits the glycopeptide bondbetween asparagine and glucosamine) and 5) after enzymatic treatment,wherein the 35S label has been removed from the 45,000 molecular weightspecies.

FIG. 2 shows the result of an investigation of the site of action of theinhibitor of the present invention in an enzyme system containing, inaddition to factor VII, factor X, the enzyme of coagulation which, whenactivated by the complex tissue factor-factor VII, converts prothrombinto thrombin, thereby leading to the formation of the fibrin clot. Theability of factor VII to convert factor X to its active form aftercomplexation with tissue factor was estimated by following thedegradation of a chromogenic substrate specific for activated factor X,or more directly by following the formation of activated factor X.Factor X activation by the complex tissue factor/factor VII yields aproteolytic fragment and activated factor X. The activation process wasfollowed by immunodetection of the product formed in the presence and inthe absence of the endothelial cell inhibitor.

Since in the presence of the inhibitor there is no detectable formationof activated factor X, the block of the pathway must occur at the levelof factor VII, or perhaps the inhibition affects tissue factor directly.

As shown in FIG. 3A, increasing amounts of factor VII do not overridethe inhibition which is overcome only by higher concentrations of tissuefactor. FIG. 3A shows that increasing amounts of factor VII added to anincubation mixture containing the inhibitor fail to relieve the block ofthe coagulation pathway: --10 , control samples; □--□, samplescontaining inhibitor. FIG. 3B shows the same incubation mixture as inFIG. 3A but using increasing amounts of thromboplastin (tissue factor)results in the development of color from the breakdown of thechromogenic substrate by activated factor X: -- , control samples; □--□,sample containing 10 μl of inhibitor; -- , samples containing 20 μl ofinhibitor.

The site of action of this inhibitor is of special significance, sincethe complexation of tissue factor with factor VII is seen as a centralevent in the initiation of the coagulation cascade. Tissue factor-likeactivity has been detected in endothelial cells when subjected tocertain stimuli and also in malignant cells where this procoagulantactivity may enhance tumor progression.

Although the glycoproteins of the present invention can be synthesizedby endothelial cells, the glycoproteins of the present invention canalso be obtained by genetic engineering methods which are well known tothose skilled in the art.

The present invention further includes DNA molecules comprising thenucleotide sequence coding for the tissue factor inhibiting protein ofthe invention, replicable expression vehicles containing said DNAmolecules, hosts transformed therewith, and the tissue factor inhibitoryprotein produced by expression of such transformed hosts. The term "DNAmolecules" includes genomic DNA, cDNA, synthetic DNA, and combinationsthereof.

The cloning of the tissue factor inhibiting protein of the presentinvention may be carried out by a variety of techniques. According toone approach, specific antibodies (polyclonal or monoclonal) to thetissue factor inhibiting protein are produced and used to clone thetissue factor inhibiting protein cDNA. This approach includes thefollowing three steps:

a. Preparation of Antibodies

The antibodies to the tissue factor inhibiting protein of the presentinvention can be produced either by using the substantially purifiedtissue factor inhibiting protein of the present invention or by usingone or more synthetic peptides identical to the known sequence of theprotein, e.g., the N-terminal protein sequence, or by fusing one of thepossible nucleotide sequences deduced from the amino acid sequence ofthe tissue factor inhibiting protein to the gene coding for Protein Aand expressing the fused Protein A-tissue factor inhibiting protein inE. coli.

For obtaining polyclonal antibodies, the substantially purified tissuefactor inhibiting protein or the synthetic peptides linked to a carrierprotein are injected into a mammal. For the production of monoclonalantibodies, the fused Protein A-tissue factor inhibiting proteinsynthetic gene is expressed in E-coli, the fused protein obtained ispurified by affinity chromatography on IgG Sepharose column, andinjected into mice. Alternatively, the substantially purified tissuefactor inhibiting protein of the present invention is injected intomice.

b. Screening of Tissue Factor Inhibiting Protein Producing Cells

Antibodies to the tissue factor inhibiting protein are used to searchfor cells producing the tissue factor inhibiting protein byimmunofluorescence or by Western blot.

c. Preparation of cDNA from Producing Cells

mRNA is extracted from the tissue factor inhibiting protein producingcells and eDNA is prepared using reversed transcriptase. The cDNA iscloned in an expression vector such as λ gT 11 and screened by the useof antibodies. The λ gT 11 expression vector can be used for insertionof DNA up to 7 kb in length at a unique EcoRI site 53 bases upstreamfrom the β-galactosidase termination codon. Therefore, foreign sequenceDNA may be inserted into this site and expressed under appropriateconditions as fusion protein. The λ gT 11 expression vector isparticularly useful for the construction of cDNA libraries to bescreened with antibody probes.

Following another approach, synthetic oligonucleotides or a mixture ofsynthetic oligonucleotides, whose sequence is derived from the sequenceof the protein of the present invention, e.g., the N-terminal amino acidsequence of the tissue factor inhibiting protein, are produced. Thisoligonucleotide or the mixture of oligonucleotides are used as a probefor cloning the cDNA or the genomic DNA coding for tissue inhibitingprotein.

The genomic DNA may optionally include naturally occurring introns. Itmay be obtained, for example, by extraction from suitable cells andpurification by means well known in the art. Suitable DNA preparations,such as human genomic DNA, are enzymatically cleaved by restrictionenzymes, or randomly sheared, and the fragments inserted intoappropriate recombinant vectors to form a gene library. These vectorscan then be screened with synthetic oligonucleotide probes in order toidentify a sequence coding for the tissue factor inhibiting protein ofthe invention.

Alternatively, mRNA is isolated from a cell which expresses the proteinof the present invention and is used to produce cDNA by means well knownin the art. This cDNA, after conversion to the double-stranded form, maybe cloned and the resulting clone screened with an appropriate probe forcDNA coding for the desired sequences. Once the desired clone has beenisolated, the cDNA may be manipulated in substantially the same manneras the genomic DNA. However, with cDNA there will be no introns orintervening sequences.

In order to synthesize the oligonucleotides to be used as probes, it ispossible either to perform sequence analysis of the intact tissue factorinhibiting protein or to obtain peptide fragments thereof and tocharacterize their amino acid sequence. In order to obtain peptidefragments, purified protein preparations are subjected to fragmentation,e.g., by digestion with proteases such as trypsin, chymotrypsin, orpapain by methods well known in the art (Oike et al., J. Biol. Chem.257: 9751-9758, 1982). The peptide fragments produced by digestion areseparated by reverse phase HPLC and sequenced by automatic amino acidsequencing techniques.

As described above, the sequence corresponding to the amino acids at theN-terminal portion of the protein was determined as follows:

NH₂-X-X-Glu-Glu-Asp-Glu-Glu-Phe-Thr-X-Ile-Thr-Asp-Ile-Lys-Pro-Pro-Leu-Gln-Lys-Pro-Thr-

Once one or more suitable peptide fragments have been sequenced, or apartial sequence of the protein is determined, the DNA sequences capableof encoding them are examined. Because of the degeneration of thegenetic code, more than one codon may be used to encode a particularamino acid, and one or more different oligonucleotides can be produced,each of which would be capable of encoding the tissue factor inhibitingprotein peptide fragments [cf. Watson, in Molecular Biology of the Gene,3rd Edition, W. A. Benjamin, Inc., Menlo Park, Calif. (1977), pp.356-357]. However, only one member of the set contains the nucleotidesequence that is identical to the nucleotide sequence of the gene. Itspresence within the set and its capability to hybridize to DNA even inthe presence of the other members of the set, makes it possible to usethe unfractionated set of oligonucleotides in the same manner in whichone would employ a single oligonucleotide to clone the gene that encodesthe peptide. The use of such oligonucleotide or set of oligonucleotidescontaining the theoretical "most probable" sequence capable of encodingthe tissue factor inhibiting protein gene fragments, following the"codon usage rules" disclosed by Lathe et al. in J. Molec. Biol.183:1-12, 1985, permits one to identify the sequence of a complementaryoligonucleotide or set of oligonucleotides which is capable ofhybridizing to the "most probable" sequence encoding the tissue factorinhibiting protein or at least a portion thereof, or a set of suchsequences. This oligonucleotide containing such a complementary sequencemay then be synthesized and used as a probe to identify and isolate thegene of the tissue factor inhibiting protein of the invention.

Once a suitable oligonucleotide, or set of oligonucleotides, which iscapable of encoding a fragment of the tissue factor inhibiting proteingene, or which is complementary to such an oligonucleotide, or set ofoligonucleotides, is identified using the abovedescribed procedure, itis synthesized and hybridized to a DNA or, preferably, to a cDNApreparation derived from cells which are capable of expressing thedesired gene, preferably after the source of cDNA has been enriched forthe desired sequences, e.g., by extracting RNA from cells which producehigh levels of the desired gene and then converting it to thecorresponding cDNA by using the enzyme reverse transcriptase.

Procedures for hybridization of nucleic acids are common knowledge, andare disclosed, for example, in Maniatis, Molecular Cloning: A LaboratoryManual, and Haymes et al., Nucleic Acid Hybridization: A practicalApproach, IRL Press, Oxford, England (1985). By hybridization with theabove nucleotide or set of oligonucleotide probes, it is possible toidentify in a cDNA or genomic library the DNA sequences capable of suchhybridization, which sequences are then analyzed to determine to whatextent they contain encoding sequences for the tissue factor inhibitingprotein of the present invention.

By the same or similar techniques, it has been possible to successfullyclone the genes for several human proteins, such as the tissue-typeplasminogen activator, cf. Pennica et al., Nature 301: 214-221, 1983.

The DNA molecules coding for the tissue factor inhibiting factor of thepresent invention obtained by the above-described methods are theninserted into appropriately constructed expression vectors by techniqueswell known in the art. Double-stranded cDNA is linked to plasmid vectorsby homopolymeric tailing or by restriction linking involving the use ofsynthetic DNA linkers or blunt-ended ligation techniques. DNA ligasesare used to ligate the DNA molecules and undesirable joining is avoidedby treatment with alkaline phosphatases.

In order to be capable of expressing a desired protein, an expressionvector should comprise also specific nucleotide sequences containingtranscriptional and translational regulatory information linked to theDNA coding for the desired protein in such a way as to permit geneexpression and production of the protein. First, in order for the geneto be transcribed, it must be preceded by a promoter recognizable by RNApolymerase, to which the polymerase binds and thus initiates thetranscription process. There are a variety of such promoters in use,which work with different efficiencies (strong and weak promoters). Theyare different for prokaryotic and eukaryotic cells.

The promoters that can be used in the present invention may be eitherconstitutive, for example, the in promoter of bacteriophage λ the bispromoter of the β-lactamase gene of pBR322, and the CAT promoter of thechloramphenicol acetyl transferase gene of pPR324, etc., or inducible, msuch as the prokaryotic promoters including the major right and leftpromoters of bacteriophage λ Pz and Pm), the trp, recA, lacZ, lacI,ompF, and gal promoters of E. coli, or the trp-lac hybrid promoter, etc.(Glick, J. Ind. Microbiol. 1: 277-282, 1987).

Besides the use of strong promoters to generate large quantities ofmRNA, in order to achieve high levels of gene expression in prokaryoticcells, it is necessary to use also ribosome-binding sites to ensure thatthe mRNA is efficiently translated. One example is the Shine-Dalgarnosequence (SD sequence) appropriately positioned fomr the initiationcodon and complementary to the 3'-terminal sequence of 16S RNA.

For eukaryotic hosts, different transcriptional and translationalregulatory sequences may be used, depending upon the nature of the host.They may be derived from viral sources, such as adenovirus, bovinepapilloma virus, Simian virus, or the like, where the regulatory signalsare associated with a particular gene which has a high level ofexpression. Examples are the TK promoter of Herpes virus, the SV40 earlypromoter, the yeast ga14 gene promoter, etc. Transcriptional initiationregulatory signals may be selected which allow for repression andactivation, so that expression of the genes can be modulated.

The DNA molecule comprising the nucleotide sequence coding for thetissue factor inhibiting protein of the invention and the operablylinked transcriptional and translational regulatory signals is insertedinto a vector which is capable of integrating the desired gene sequencesinto the host cell chromosome. The cells which have stably integratedthe introduced DNA into their chromosomes can be selected by alsointroducing one or more markers which allow for selection of host cellswhich contain the expression vector. The marker may provide forphototrophy to an auxotropic host, biocide resistance, e.g.,antibiotics, or heavy metals, such as copper or the like. The selectablemarker gene can either be directly linked to the DNA gene sequences tobe expressed, or may be introduced into the same cell byco-transfection. Additional elements may also be needed for optimalsynthesis of single chain binding protein mRNA. These elements mayinclude splice signals, as well as transcription promoters, enhancers,and termination signals. cDNA expression vectors incorporating suchelements include those described by Okayama in Mol. Cel. Biol. 3: 280,1983.

In a preferred embodiment, the introduced DNA molecule will beincorporated into a plasmid or vital vector capable of autonomousreplication in the recipient host. Factors of importance in selecting aparticular plasmid or viral vector include the ease with which therecipient cells that contain the vector may be recognized and selectedfrom those recipient cells which do not contain the vector; the numberof copies of the vector which are desired in a particular host; andwhether it is desirable to be able to "shuttle" the vector between hostcells of different species.

Preferred prokaryotic vectors include plasmids such as those capable ofreplication in E. coli, such as pBR322, Co1E1, pSC101, pACYC 184, etc.,(see Maniatis et al., Molecular Cloning: A Laboratory Manual); Bacillusplasmids such as pC194, pC221, pT127, etc. (see Gryzcan, The MolecularBiology of the Bacilli, Academic Press, NY, 1982, pp 307-329);Streptomyces plasmids including pIJ101 (cf. Kendall et al., J.Bacteriol. 169:4177-4183, 1987); Streptomyces bacteriophages such as0C31 (Chater et al., in Sixth International Symposium onActinomycetales. Biology, Akademiai Kaido, Budapest, Hungary, 1986, pp.46-54)' and Pseudomonas plasmids (John et al., Rev. Infect. Dis. 8:693-704; and Izaki, Jpn. J. Bacteriol. 33: 729-742, 1978).

Preferred eukaryotic plasmids include BPV, vaccinia, SV40, 2-microncircle, etc., or their derivatives. Such plasmids are well known in theart.

Once the vector or DNA sequence containing the constructs has beenprepared for expression, the DNA constructs may be introduced into anappropriate host cell by any of a variety of suitable means:transformation, transfection, conjugation, protoplast fusion,electroporation, calcium phosphate-precipitation, direct microinjection,etc.

Host cells to be used in this invention may be either prokaryotic oreukaryotic. Preferred prokaryotic hosts include bacteria such as E.coli, Bacillus, Streptomyces, Pseudomonas, Salmonella, Serratia, etc.The most preferred prokaryotic host is E. coli. Bacterial hosts ofparticular interest include E. coli K12 strain 294 (ATCC 31446), E. coliX1776 (ATCC 31537), E. coli W3110 (F-, lambda-, prototropic (ATCC27325)), and other enterobacteria such as Salmonella typhimurium orSerfaria marcescens and various Pseudomonas species. Under suchconditions, the protein will not be glycosylated. The prokaryotic hostmust be compatible with the replicon and control sequences in theexpression plasmid.

Preferred eukaryotic hosts are mammalian cells, such as human, monkey,mouse, and Chinese hamster ovary (CHO) cells, because they providepost-translational modifications to protein molecules including correctfolding or glycosylation at correct sites. Also, yeast cells can carryout post-translational peptide modification including glycosylation. Anumber of recombinant DNA strategies exist which utilize strong promotersequences and high copy number of plasmids which can be utilized forproduction of the desired proteins in yeast. Yeast recognizes leadersequences on cloned mammalian gene products and secretes peptidesbearing leader sequences, i.e., pre-peptides.

After introduction of the vector, the host cells are grown in aselective medium, which selects for the growth of vector-containingcells. Expression of the cloned gene sequence(s) results in theproduction of the desired tissue factor inhibiting protein or a fragmentthereof. The expressed protein is then isolated and purified inaccordance with any purification method including extraction,precipitation, chromatography, electrophoresis, or the like.

A further purification procedure that may be used in preference forpurifying the protein of the invention is affinity chromatography. Forthis purpose, polyclonal or monoclonal antibodies to the tissue factorinhibiting protein are produced and immobilized on a gel matrixcontained within a column. Impure preparations containing therecombinant protein are passed through the column. The protein will bebound to the column by the specific antibody while the impurities willpass through. After washing, the protein is eluted from the gel by achange in pH or ionic strength.

The monoclonal antibodies used in the present invention can be preparedusing conventional hybridoma techniques, such as disclosed in Kohler etal., Nature 256:495, 1975; Kohler et al., Eur. J. Immunol 6:511, 1976.In general, such procedures involve immunizing an animal with thedesired purified protein antigen or with a synthetic peptide having theN-terminal sequence of the desired protein conjugated to a suitablecarrier, such as bovine serum albumin. Spleen cells of such animals areisolated and fused with a suitable myeloma cell line. After fusion, theresulting hybridoma cells are selectively maintained in HAT medium andthen cloned. The hybridoma cells obtained through such a selection arethen assayed to identify clones which secrete antibodies capable ofbinding the tissue factor inhibiting protein. After identification, thedesired clone can be grown in bulk, either in suspension culture or inascitic fluid by injecting the cells into the peritoneum of suitablehost mice.

The monoclonal antibodies produced by said hybridomas, afterpurification and immobilization, are very efficient for the purificationof the tissue factor inhibiting protein in affinity purificationprocedures using an immunoadsorbent column.

Additionally, the monoclonal antibodies prepared with the tissue factorinhibiting protein of the present invention can be labelled byconventional means and used in assays for the proteins of the presentinvention. These immunological assays are well known to those skilled inthe art, and are exemplified by U.S. Pat. No. 4,376,110 to David et al.,which patent is hereby incorporated by reference.

Briefly, the immunometric assays that can be performed with eitherpolyclonal or monoclonal antibodies of the present invention can be usedto determine the presence and/or concentration of an antigenic substancein a fluid. The assays involve contacting a sample of the fluid with ameasured amount of a labelled antibody according to the presentinvention, and measuring the amount of labelled antibody associated withthe antigen. The amount of labelled antibody measured is then related tothe amount of labelled antibody measured for a control sample preparedas above, the control sample being known to be free of the antigenicsubstance, to determine the presence of antigenic substance in the fluidsample, or relating the amount of labelled antibody measured with theamount of labelled antibody measured for samples containing knownamounts of antigenie substances prepared as above, to determine theconcentration of antigenic substances in the fluid sample.Alternatively, the antigen can be labelled, and the amount brought downcan be measured against a standard curve.

The labels for the antibodies can be selected from among the knownlabels for immunometric assays, e.g., radiolabels, enzyme labels,chemiluminescent labels, fluorescent labels, and the like.

It should be understood that the present invention comprehends tissuefactor inhibiting proteins which are effective in inhibiting theinitiation of the coagulation process which is promoted by tissuefactor.

It should be further understood that the decoyants of the presentinvention can be modified by extending the polypeptide of the presentinvention or by adding specific chemical moieties intended to aid indrug design or to permit the tissue factor inhibitors to be used foradditional purposes. One such modification would be to extend thepolypeptide by moieties intended to affect solubility, e.g., by theaddition of a hydrophilic residue, such as serine, or a charged residue,such as glutamic acid. Furthermore, the protein could be extended forthe purpose of stabilization and preservation of a desired conformation,such as by adding cysteine residues for the formation of disulfidebridges.

The tissue factor inhibitors may also be modified to make themdetectable after administration, such as by radioiodination with aradioactive iodine isotope, directly, or by adding tyrosine forsubsequent radioiodination.

Further, alterations of the hemostatic system resulting in an increasedincidence of thrombotic disorders is a frequent consequence ofneoplasia.

The inhibitors of the present invention are useful in inhibitingintravascular clotting and preventing the formation of fibrin clots bothin vitro and in vivo. The inhibitors of the present invention areparticularly useful for anticoagulant therapy in prophylaxis of venousthrombosis and as treatment for preventing its extension, as well as toprovide a low-dose regiment for prevention of postoperative deep venousthrombosis and pulmonary embolism in patients undergoing majorabdominothoracic surgery, particularly those who are at risk ofdeveloping thromboembolic disease. The compounds of the presentinvention can also be used for the propylaxis and treatment of pulmonaryembolism and atrial fibrillation with embolization. Additionally, thecompounds of the present invention can be used to prevention of clottingin arterial and heart surgery as well as for prevention of cerebralthrombosis in evolving stroke. The compounds of the present inventioncan be used as an adjunct both in treating coronary occlusion with acutemyocardial infarction and in the propylaxis and treatment of peripheralarterial embolism. The compounds can also be employed as ananticoagulant in blood transfusions, extracorporeal circulation, anddialysis procedures and in blood samples for laboratory purposes.

One skilled in the art can determine the amount of the inhibitors of thepresent invention to be administered, particularly in therapeutic ratherthan preventative amounts. When the compounds are administered intherapeutic amounts, the dosage should be regulated by frequent bloodcoagulation tests. If the coagulation test is unduly prolonged, or ifthere is any indication of hemorrhage, use of the inhibitor would bediscontinued.

The inhibitors of the present invention can be administered incompositions containing the active ingredient in a pharmaceuticallyacceptable carrier. Determination of the effective amounts is within theskill of the art.

In addition to the inhibitors of the present invention, thesepharmaceutical compositions may contain suitable pharmaceuticallyacceptable carriers comprising excipients and auxiliaries whichfacilitate processing of the active compounds into preparations whichcan be used pharmaceutically. Preferably, the preparations contain fromabout 0.1 to about 99 percent of active compound, together with theexcipient.

The pharmaceutical preparations of the present invention can bemanufactured in a manner which is itself known, for example, by means ofconventional mixing, dissolving, or lypophilizing processes. Suitableformulations for parenteral administration include aqueous solutions ofthe active compounds in water-soluble form. In addition, suspensions ofthe active compounds as appropriate oily injection suspensions may beadministered. Suitable lipohilic solvent or vehicles include fatty oilssuch as sesame oil, or synthetic fatty acid esters such as ethyl oleateor triglycerides. Aqueous injection suspensions may contain substanceswhich increase the viscosity of the suspension such as sodiumcarboxymethyl cellulose, sorbitol, and/or dextran. Optionally, thesuspension may also contain stabilizers. The tissue factor inhibitors ofthe present invention may also be administered in the form of liposomes,pharmaceutical compositions in which the active ingredient is containedeither dispersed or variously present in corpuscles consisting ofaqueous concentric layers adherent to lipidic layers. The activeingredient may be present both in the aqueous layer and in the lipidiclayer, or, in any event, in the non-homogeneous system generally knownas a liposomic suspension.

Possible pharmaceutical preparations which can be used rectally include,for example, suppositories, which consist of a combination of the activecompounds with a suppository base. Suitable suppository bases are, forexample, natural or synthetic triglycerides, paraffin hydrocarbons,polyethylene glycols, or higher alkanols. In addition, it is alsopossible to use gelatin rectal capsules which consist of a combinationof the active compounds with a base. Possible base materials include,for example, liquid triglycerides, polyethylene glycols, or paraffinhydrocarbons.

Suitable formulations for parenteral administration include aqueoussolutions of the active compounds in water-soluble form, for example, aswater-soluble salts. In addition, suspension of the active compounds asappropriate oily injection suspensions may be administered. Suitablelipophilic solvents or vehicles include fatty oils, for example, sesameoil, or synthetic fatty acid esters, such as ethyl oleate ortriglycerides.

Aqueous injection suspensions may contain substances which increase theviscosity of the suspension such as sodium carboxymethyl cellulose,sorbitol, and/or dextran. Optionally, the suspension may also containstabilizers.

The compounds of the present invention may be immobilized to affinitymatrices and biological molecules by well established procedures. Forexample, these compounds may be immobilized to an affinity matrix bytreatment of such a mixture with dicyclohexylcarboi-imide in a suitablesolvent, or by glutaraldehyde crosslinking.

The compounds of the present invention can also be administered in theform of liposomes, pharmaceutical compositions in which the activeingredient is contained either dispersed or variously present incorpuscles consisting of aqueous concentric layers adherent to lipidiclayers (hydrophobic). The drug may be present both in the aqueous layerand in the lipidic one (inside or outside) or, in any event, in thenon-homogeneous system generally known as a liposomic suspension.

The inhibitor of the present invention can be administered to a patientat risk for clot formation in effective amounts to prevent initiation ofthe clotting mechanism. Alternatively, the inhibitors of the presentinvention can be used therapeutically to treat coronary occlusion withacute myocardial infarction and peripheral arterial embolism. Generally,amounts ranging from about 1 mg to about 12 mg a day can be administeredto prevent clotting, and preferably from about 4 to about 6 mg per day.Effective amounts to be administered are dependent upon the individualpatient, and can readily be determined by one skilled in the art.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingcurrent knowledge, readily modify and/or adapt for various applicationssuch specific embodiments without departing from the generic concept,and, therefore, such adaptations and modifications should and areintended to be comprehended within the meaning and range of equivalentsof the disclosed embodiments. It is to be understood that thephraseology or terminology employed herein is for the purpose ofdescription and not of limitation.

What is claimed is:
 1. A purified antibody having an antibody bindingregion specific to an epitope of a purified and isolated tissue factorinhibiting protein, said protein comprising a sulfated glycoproteinhaving a molecular weight of about 45 kDa, wherein(a) said protein hasthe ability to inhibit the activity of tissue factor; and (b) saidglycoprotein includes the amino acid sequence:X₁ -X₂-Glu-Glu-Asp-Glu-Glu-Phe-Thr-X₃-Ile-Thr-Asp-Ile-Lys-Pro-Pro-Leu-Gln-Lys-Pro-Thr, where X₁, X₂, and X₃can be the same or different and denote any amino acid.
 2. An antibodyaccording to claim 1, wherein said tissue factor inhibiting protein isin the form of a salt.
 3. An antibody according to claim 1, wherein saidtissue factor inhibiting protein is a recombinant protein.
 4. A methodfor assaying for tissue factor inactivating protein comprisingcontacting a sample fluid with a labelled antibody according to claim 22to form a conjugate of the tissue factor inhibiting protein and theantibody, and assaying the conjugate to determine the presence andamount of the tissue factor inhibiting protein.
 5. The method accordingto claim 4 wherein the label is a radioactive label.
 6. The methodaccording to claim 4 wherein the label is an enzyme label.
 7. The methodaccording to claim 4 wherein the label is a chemiluminescent label. 8.The method according to claim 4 wherein the label is a fluorescentlabel.
 9. The method of claim 4 wherein the antibody is a monoclonalantibody.